Methods for control of flux in metabolic pathways

ABSTRACT

The invention pertains to a method for preparing cells that can be used as biocatalysts by inducing in them a growth-decoupled state, in which interferase inhibits the expression of genes except the ones that code for the pathway enzymes of interest. mRNAs that code for interferase-resistant products are overexpressed in the background of a metabolically-frozen cell. Enzymes that compete for a substrate or product of the pathway of interest may be altered such that the enzyme is sensitive to a site-specific protease, which protease is inducible in the host cell.

RELATED APPLICATIONS SECTION

This application claims the benefit under 35 U.S.C. §119(e) of U.S. application Ser. No. 13/132,721, filed Jul. 12, 2011, which is a national stage filing under 35 U.S.C. §371 of international application number PCT/US2009/067841, filed Dec. 14, 2009, which was published under PCT Article 21(2) in English and claims the benefit under 35 U.S.C. §119(e) of U.S. provisional application Ser. No. 61/201,783, filed Dec. 15, 2008, each of which is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Metabolic engineering has allowed production of chemicals of commercial interest through manipulation of biochemical reactions in the cell. However, all organisms have evolved with the objective of replicating their genetic material and, therefore, production of chemicals that may be of commercial interest may conflict with essential cellular goals. For example, diversion of nutrients and energy for the production of a compound may result in a shortage of those substrates for production of biomass. The organism that is engineered may either evolve away from producing the compound of interest or grow sub-optimally.

To address this issue, systems have been engineered for stationary phase-associated production of a compound of interest. However, a limitation is that stationary phase itself elicits a series of responses that are aimed at protecting the cell during non-growing conditions, so cellular resources are still needed for combating stress and preserving the stationary-phase phenotype. This effect may be pronounced when the product of interest is itself toxic or induces a stress response.

Recently, the development of cell-free systems has allowed for the in vitro production of proteins (for a review see Swartz (2006) J Ind Microbiol Biotechnol 33:476-85). A cell-free platform may also be used to produce metabolites of interest through coordinated expression of proteins in a pathway. The procedure entails growing a biomass of cells, opening the cells to liberate the cytoplasmic components, removing the genomic DNA, and using the genome-free machinery to produce a user-specified set of enzymes to serve as biocatalysts. This presents a unique opportunity for producing an environment that resembles stationary phase production systems but free of stress or other responses, although a limitation may be relatively slow kinetics of the set of reactions, given that diffusion of intermediates from one enzyme into the next is needed for a pathway to work.

PUBLICATIONS

-   International Patent Application no. WO/2007/137144; Inouye (2006) J     Cell Physiol 209:670-6; Suzuki et al. (2007) Nat Protoc 2:1802-10;     Suzuki et al. (2005) Mol Cell 18:253-61.

SUMMARY OF THE INVENTION

Compositions and methods are provided for controlling metabolic pathway flux through manipulation of targeted enzymes involved in a pathway of interest, including manipulation to maintain or alter the cellular concentration of key pathway enzymes during a cell growth phase, followed by manipulation to (a) increase concentrations of key pathway enzymes and/or (b) decrease concentrations of competitive enzymes during a production phase, where the product of the pathway of interest is produced. The cell growth phase necessarily involves intact cells, while the production phase is may be performed with intact cells or lysates of such cells.

Specifically, the microbial cells are genetically modified, such that mRNA encoding enzymes involved in the pathway of interest are resistant to the ribonuclease activity of interferases, also referred to as TA toxins. After induction of interferase expression, the microbial cells substantially lack new polypeptide synthetic activity, except for enzymes involved in a pathway of interest, referred to herein as cells in a metabolically frozen state. The microbial cells may be prokaryotic or eukaryotic.

Generally a plurality of enzyme coding sequences in a single pathway are modified, usually at least two coding sequences, at least three coding sequences, or more. Generally one or more of the modified sequences control flux through the pathway, e.g. by catalyzing an initial diversion of a central metabolite to a pathway of interest, by catalyzing a rate-limiting step of the pathway, by controlling the production of an input substrate to the pahway, etc. Such modified coding sequence may be referred to as interferase resistant pathway enzyme sequences.

In some embodiments the microbial cells express endogenous interferase genes. In other embodiments, interferase coding sequences are introduced into the microbial cells. Interferase coding sequences may be obtained, for example, from sources such as Mycobacterium tuberculosis and Myxococcus xanthus. A metabolically-frozen state is induced by one or both of (a) expression of TA toxins, usually exogenous TA toxins; and (b) attenuation of TA antitoxins.

In some embodiments of the invention, expression of the plurality of enzymes in the pathway of interest is inducible. In such embodiments, the induction of a metabolically frozen state may be coordinated with induction of expression of the plurality of enzymes in the pathway of interest. Following induction, the cell continues to produce only the enzymes in the pathway of interest, providing a benefit for the synthesis of metabolites of the pathway. Only the set of desired enzymes continues to be produced, but without the limitation of stress responses to the cell and providing substrate channeling through the pathway by the close proximity of enzymes in the cell.

Optionally, in some embodiments of the invention, one or more enzymes that compete for substrates of enzymes in the pathway of interest are genetically altered to comprise at least one cleavage site for a site-specific protease. Generally in such embodiments, a further genetic modification is made such that the cell expresses an interferase resistant form of the cognate site-specific protease. In some embodiments, expression of the cognate site-specific protease is coordinately induced with expression of the enzymes of the pathway of interest.

In some embodiments, methods are provided for producing a product of interest at a high flux rate, the method comprising: growing cells that are genetically modified to coordinately induce interferase activity, which substantially ablates new polypeptide synthesis by cleavage of mRNA, and interferase resistant enzymes in a pathway of interest to a desired density; inducing interferase activity and expression of the interferase resistant enzymes; lysing the cells; and producing the product of the pathway in a cell-free system comprising the lysate. Additional substrate, nutrients, cofactors, buffers, reducing agents, ATP generating systems, etc. may be added to the cell-free system. In an alternative embodiment the cells are grown to a desired density; interferase activity and expression of the interferase resistant enzymes is induced; and the product of the pathway is produced in a cellular environment.

In other embodiments, the genetically modified cell is provided. In another embodiment, lysates of such a genetically modified cell are provided, which lysate may be combined with one or more of substrate, nutrients, cofactors, buffers, reducing agents, ATP generating systems, etc. to generate a cell-free system for producing a product of interest.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C provide a metabolic scheme A for production of shikimic acid.

FIGS. 2A-2C provide a metabolic scheme B for production of shikimic acid.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications (published or unpublished), and other publications referred to herein are incorporated by reference in their entireties. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are incorporated herein by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

Citation of publications or documents is not intended as an admission that any of such publications or documents are pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

As used herein, “a” or “an” means “at least one” or “one or more” unless otherwise indicated.

The nucleic acids used to practice this invention, whether RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof, may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.

Host cells of interest for pathway engineering include a wide variety of heterotrophic and autotrophic microorganisms, including bacteria, fungi and protozoans. Species of interest include, without limitation, S. cerevisiae, E. coli, Picchia pastoris, etc.

Techniques for the manipulation of nucleic acids, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Flux.

The term “flux” as used herein refers to the rate that molecules pass through a pathway or reaction of interest. Among the factors that control flux are rate of catalysis of enzymes in the pathway, the availability of substrate, the concentration of enzymes in a cell, the proximity of enzymes in a pathway, etc.

While a high rate of flux through a pathway of interest is desirable, at the same time it can create toxicity issues if a product not normally accumulated at high levels in the cell is produced at a high rate. A stressed cell produces a number of proteins undesirable for maintaining active biocatalysis, such as nucleases, heat shock proteins, proteases and the like.

The methods of the invention provide a means of controlling flux through a pathway, such that a healthy cell, with substantially normal physiology, can be grown to high density, following which the concentration of enzymes involved in a pathway is modulated to result in an increase in the pathway flux, and accumulation of a desired product. Modulation of enzyme concentration is accomplished by inducing interferase expression, while protecting the mRNA of key pathway enzymes from degradation by the interferase. In this manner the key pathway enzymes are increased in concentration, while stress response genes are not expressed. In combination or as an alternative approach, competitive enzymes that reduce pathway flux or accumulation of the desired product may be engineered to contain a sequence specific protease cleavage site, where expression of the protease is induced in the cells at a selected time.

Methods of determining flux rates are known and used in the art, for example as described by Wiechert et al. (2001) Metab. Eng. 3, 265-283, A universal framework for 13C metabolic flux analysis”, and Metab Eng. 2001 July; 3(3):195-206; or metabolic engineering texts such as Lee and Papoutsakis, 1999, Stephanopoulos, Aristidou, Nielsen, 1998, Nielsen and Eggeling, 2001, each herein specifically incorporated by reference. Flux may be calculated from measurable quantities using techniques such as metabolic flux analysis (MFA), for example by direct measurement of the conversion of isotopically labeled substrate.

Enzyme Pathway:

As used herein, the term “enzyme pathway” or “pathway of interest” refers to a cellular system for converting a substrate to a product of interest, where the system comprises a plurality of enzymes and may additionally comprise substrates acted upon by one or more of the enzymes, products of the enzyme-catalyzed reaction, co-factors utilized by the enzymes, and the like. The system may be present in an intact cell, or in a lysate of a cell. Many metabolic pathways are known and have been described in microbial systems, and are accessible in public databases. For example, a number of reference books are available, including, inter alia, The Metabolic Pathway Engineering Handbook (2009), ed. C. Smolke, CRC, ISBN-10: 1420077651 and 1439802963; Metabolic Engineering: Principles and Methodologies (1998) Stephanopoulos, Academic Press ISBN-10: 0126662606, Greenberg DM. Metabolic Pathways: Energetics, tricarboxylic acid cycle, and carbohydrates. Academic Press; 1967; Greenberg M. Metabolic pathways. Academic Press; 1968; Greenberg DM. Metabolic pathways. Academic; 1970; and Greenberg D M, Vogel H J. Metabolic pathways. Academic; 1971, each herein specifically incorporated by reference.

Pathways of interest include, without limitation, pathways involved in carbohydrate, amino acid, nucleic acid, steroid, and fatty acid metabolism, and may include synthesis of antibiotics, e.g. actinomycin, bleomycin, rifamycin, chloramphenicol, tetracycline, lincomycin, erythromycin, streptomycin, cyclohexamide, puromycin, cycloserine, bacitracin, penicillin, cephalosporin, vancomycin, polymyxin, and gramicidin; biosurfactants e.g. rhamnolipids, sophorolipids, glycolipids, and lipopeptides; biological fuels e.g. bioethanol, biodiesel, and biobutanol; amino acids e.g. L-glutamate, L-lysine, L-phenylalanine, L-aspartic acid, L-isoleucine, L-valine, L-tryptophan, L-proline (hydroxyproline), L-threonine, L-methionine, and D-p-hydroxyphenylglycine; organic acids e.g. citric acid, lactic acid, gluconic acid, acetic acid, propionic acid, succinic acid, fumaric acid, and itaconic acid; fatty acids e.g. arachidonic acid, polyunsaturated fatty acid (PUBA), and γ-linoleic acid; polyols e.g. glycerol, mannitol, erythritol, and xylitol; flavors and fragrances e.g. vanillin, benzaldehyde, dixydroxyacetone, 4-(R)-decanolide, and 2-actyl-1-pyrroline; nucleotides e.g. 5′-guanylic acid and 5′-inosinic acid; vitamins e.g. vitamin C, vitamin F, vitamin B2, provitamin D2, vitamin B12, folic acid, nicotinamide, biotin, 2-keto-L-gulonic acid, and provitamin Q10; pigments e.g. astaxathin, β-carotene, leucopene, monascorubrin, and rubropunctatin; sugars and polysaccharides e.g. ribose, sorbose, xanthan, gellan, and dextran; biopolymers and plastics e.g. polyhydroxyalkanoates (PHA), poly-γ-glutamic acid, and 1,3-propanediol; and the like as known in the art.

A number of reactions may be catalyzed by enzymes in pathways of interest. Broad classes, which can be identified by enzyme classification number, provided in parentheses, include (EC 1) oxidoreductases, e.g. dehydrogenases, oxidases, reductases, oxidoreductases, synthases, oxygenases, monooxygenases, dioxygenases, lipoxygenases, hydrogenases, transhydrogenases, peroxidases, catalases, epoxidases, hydroxylases, demethylases, desaturases, dismutases, hydroxyltransferases, dehalogenases, deiodinases, etc.; (EC2) transferases, e.g. Transaminases, kinases, dikinases, methyltransferases, hydroxymethyltransferases, formyltransferases, form iminotransferases, carboxytransferases, carbamoyltransferases, amidinotransferases, transaldolases, transketolases, acetyltransferases, acyltransferases palmitoyltransf erases, succinyltransf erases, malonyltransf erases, galloyltransf erases, sinapoyltransferases, tigloyltransferases, tetradecanoyltransferases, hydroxycin namoyltransf erases, feruloyltransf erases, mycolyltransf erases, benzoyltransf erases, piperoyltransf erases, trimethyltridecanoyltransferase, myristoyltransferases, coumaroyltransferases, thiolases, aminoacyltransferases, phosphorylases, hexosyltransferases, pentosyltransferases, sialyltransf erases, pyridinylases, diphosphorylases, cyclotransferases, sulfurylases, adenosyltransferases, carboxyvinyltransf erases, isopentenyltransferases, am inocarboxypropyltransf erases, dimethylallyltransferases, farnesyltranstransferases, hexaprenyltranstransferases, decaprenylcistransferases, pentaprenyltranstransferases, nonaprenyltransf erases, geranylgeranyltransferases, aminocarboxypropyltransferases, oximinotransf erases, purinetransferases, phosphodismutases, phosphotransferases, nucleotidyltransferases, polymerases, cholinephosphotransferases, phosphorylmutases, sulfurtransferases, sulfotransferases, CoA-transferases, etc.; (EC3) hydrolases, e.g. lipases, esterases, amylases, peptidases, hydrolases, lactonases, deacylases, deacetylases, pheophorbidases, depolymerases, thiolesterases, phosphatases, diphosphatases, triphosphatases, nucleotidases, phytases, phosphodiesterases, phospholipases, sulfatases, cyclases, oligonucleotidases, ribonucleases, exonucleases, endonucleases, glycosidases, nucleosidases, glycosylases, aminopeptidases, dipeptidases, carboxypeptidases, metallocarboxypeptidases, omega-peptidases, serine endopeptidases, cystein endopeptidases, aspartic endopeptidases, metalloendopeptidases, threonine endopeptidases, aminases, amidases, desuccinylases, deformylases, acylases, deiminases, deaminases, dihydrolases, cyclohydrolases, nitrilases, ATPases, GTPases, halidases, dehalogenases, sulfohydrolases, etc.; (EC 4) lyases, e.g. decarboxylases, carboxylases, carboxykinases, aldolases, epoxylyases, oxoacid-lyases, carbon-carbon lyases, dehydratases, hydratases, synthases, endolyases, exolyases, ammonia-lyases, amidine-lyases, amine-lyases, carbon-sulfur lyases, carbon-halide lyases, phosphorus-oxygen lyases, dehydrochlorinases, etc.; (EC 5) isomerases, e.g. isomerases, racemases, mutases, tautomerases, phosphomutases, phosphoglucomutases, aminomutases, cycloisomerase, cyclases, topoisomerases, etc.; and (EC 6) ligases, e.g. synthetases, tNRA-ligases, acid-thiol ligases, amide synthases, peptide synthases, cycloligases, carboxylases, DNA-ligases, RNA-ligases, cyclases, etc.

More specific classes include, without limitation oxidoreductases, including those (EC 1.1) acting on the CH—OH group of donors, and an acceptor; (EC 1.2) Acting on the aldehyde or oxo group of donors, and an acceptor; (EC 1.3) Acting on the CH—CH group of donors, and an acceptor; (EC 1.4) Acting on the CH—NH2 group of donors, and an acceptor; (EC 1.5) Acting on the CH—NH group of donors, and an acceptor; (EC 1.6) Acting on NADH or NADPH, and an acceptor; (EC 1.7) Acting on other nitrogenous compounds as donors, and an acceptor; (EC 1.8) Acting on a sulfur group of donors, and an acceptor; (EC 1.9) Acting on a heme group of donors, and an acceptor; (EC 1.1) Acting on diphenols and related substances as donors, and an acceptor; (EC 1.11) Acting on a peroxide as acceptor; (EC 1.12) Acting on hydrogen as donor, and an acceptor; (EC 1.13) Acting on single donors with incorporation of molecular oxygen, incorporating one or two oxygen atoms; (EC 1.14) Acting on paired donors, with incorporation or reduction of molecular oxygen, with the donor being 2-oxoglutarate, NADH, NADPH, reduced flavin, flavoprotein, pteridine, iron-sulfur protein, ascorbate, etc.; (EC 1.15) Acting on superoxide radicals as acceptor; (EC 1.16) Oxidising metal ions, and an acceptor; (EC 1.17) Acting on CH or CH2 groups, and an acceptor; (EC 1.18) Acting on iron-sulfur proteins as donors, and an acceptor; (EC 1.19) Acting on reduced flavodoxin as donor, and an acceptor; (EC 1.20) Acting on phosphorus or arsenic in donors, and an acceptor; (EC 1.21) Acting on X—H and Y—H to form an X—Y bond, and an acceptor; where acceptors for each donor category may include, without limitation: NAD, NADP, heme protein, oxygen, disulfide, quinone, an iron-sulfur protein, a flavin, a nitrogenous group, a cytochrome, dinitrogen, and H⁺.

Transferases include those: (EC 2.1) Transferring one-carbon groups; (EC 2.2) Transferring aldehyde or ketonic groups; (EC 2.3) Acyltransferases; (EC 2.4) Glycosyltransferases; (EC 2.5) Transferring alkyl or aryl groups, other than methyl groups; (EC 2.6) Transferring nitrogenous groups; (EC 2.7) Transferring phosphorus-containing groups; (EC 2.8) Transferring sulfur-containing groups; (EC 2.9) Transferring selenium-containing groups.

Hydrolases include those: (EC 3.1) Acting on ester bonds; (EC 3.2) Glycosylases; (EC 3.3) Acting on ether bonds; (EC 3.4) Acting on peptide bonds (peptidases); (EC 3.5) Acting on carbon-nitrogen bonds, other than peptide bonds; (EC 3.6) Acting on acid anhydrides; (EC 3.7) Acting on carbon-carbon bonds; (EC 3.8) Acting on halide bonds; (EC 3.9) Acting on phosphorus-nitrogen bonds; (EC 3.1) Acting on sulfur-nitrogen bonds; (EC 3.11) Acting on carbon-phosphorus bonds; (EC 3.12) Acting on sulfur-sulfur bonds; (EC 3.13) Acting on carbon-sulfur bonds.

Lyases include those: (EC 4.1) Carbon-carbon lyases; (EC 4.2) Carbon-oxygen lyases; (EC 4.3) Carbon-nitrogen lyases; (EC 4.4) Carbon-sulfur lyases; (EC 4.5) Carbon-halide lyases; (EC 4.6) Phosphorus-oxygen lyases.

Isomerases include those: (EC 5.1) Racemases and epimerases; (EC 5.2) cis-trans-Isomerases; (EC 5.3) Intramolecular isomerases; (EC 5.4) Intramolecular transferases (mutases); (EC 5.5) Intramolecular lyases.

Ligases, include those: (EC 6.1) Forming carbon-oxygen bonds; (EC 6.2) Forming carbon-sulfur bonds; (EC 6.3) Forming carbon-nitrogen bonds; (EC 6.4) Forming carbon-carbon bonds; (EC 6.5) Forming phosphoric ester bonds; (EC 6.6) Forming nitrogen-metal bonds.

Enzymes involved in a pathway may be classified according to the role of the enzymes. Direct involvement enzymes catalyze a reaction in the pathway. It is typical of pathways that such direct enzymes are one of a chain, where a product of a first enzyme is the substrate of a second, etc., which eventually results in the product of interest. Indirect involvement enzymes react in an associated pathway, usually in the production of a substrate used in the pathway. It may be a characteristic of an enzyme in these two classes that overproduction of the enzyme is toxic to the cell, even 2-fold, 3-fold or more overproduction. Such toxicity can be the result of various effects, including overproduction of a product that is toxic at high concentrations, or that the enzyme diverts resources, e.g. an initial substrate, at a rate that impacts the normal cell physiology, etc. The expression of such enzymes benefits from modulated selective increase in expression with the methods of the invention, in order to avoid undesirable stress to the cell.

Within a pathway, enzymes will vary in turnover rate and the effectiveness with which a product is produced. As a result, certain enzymes in a pathway become rate-limiting. Increasing the concentration of rate-limiting enzymes in a pathway (relative to non-rate limiting enzymes) allows increased flux through the pathway of interest. Often rate-limiting enzymes are also associated with toxicity when over-produced, and thus expression of such enzymes is desirably modulated by the methods of the invention to selectively increase expression at a selected time point.

A third class of enzymes are competing enzymes, which utilize a substrate or product of the pathway of interest. A characteristic of a competing enzyme is that the kinetics of the substrate conversion are sufficiently high that the presence of the enzyme decreases the overall yield and/or the rate of production of the desired final product catalyzed by the pathway of interest. A normal cell may require the expression of competing enzymes, and therefore rather than knocking out expression, it is desirable to selectively decrease the concentration of the enzyme by the methods of the invention, e.g. by introducing a site for a sequence-specific protease and inducing protease expression.

For convenience of naming, an enzyme in the pathway may be categorized as a first, pathway entry enzyme, or a subsequent downstream enzyme or enzymes. For convenience, the pathway entry enzyme may be referred to herein as E₁, and the downstream enzymes may be consecutively numbered, E₂, E₃, . . . E_(n). Pathways of interest for the methods of the invention will usually comprise at least two enzymes, and may comprise at least three enzymes, at least four enzymes, or more.

Enzymes in a pathway may be naturally occurring, or modified to optimize a characteristic of interest, e.g. substrate specificity, reaction kinetics, solubility, codon usage, etc. In some embodiments the complete pathway comprises enzymes from a single organism, however such is not required, and combining enzymes from multiple organisms is contemplated. For some purposes a pathway may be endogenous to the host cell, but such is also not required, and a complete pathway or components of a pathway may be introduced into a host cell. Where the system is provided in an intact cell, generally the complete set of enzymes required for pathway conversion will be present in the cell. For purposes of cell-free production, one or more enzymes may be added to the lysate so as to complete the pathway.

In the pathway system, a first substrate (S₁) is acted upon by the pathway entry enzyme, and is converted to a first product, although it will be understood by one of skill in the art that an enzyme may act upon more than one substrate simultaneously, and may produce more than one product, such that two or more pathways may be interconnected at a single enzyme. The first product is a substrate (S₂) for downstream enzyme E₂, and is converted to a second product. Depending on the complexity of the pathway, the second product may be the final product (P_(F)), or may be a substrate (S₃) for a third downstream enzyme (E₃), and is converted to a third product, which may be a substrate (S₄) for a fourth enzyme, etc. The final enzyme in the pathway, which may be E₂, E₃, E₄, etc. produces the product of interest (P_(F)). It is a characteristic of enzyme pathways that the product of one enzyme is the substrate for the next enzyme. Products may be stable or relatively labile, but in general the final product is sufficiently stable that it can be isolated from the cell or reaction mixture.

In some embodiments of the invention, the initial substrate, S₁, is a central metabolite, or cellular “commodity”. The central pathways of metabolism include glycolysis and the citric acid cycle. Such S₁ compounds are generally not specific to the pathway of interest, but are compounds widely found in various cells and are substrates for multiple enzymes and pathways. Examples of commodity substrates include, without limitation, glucose, ATP, pyruvate, phosphoenol pyruvate, and the like. A pathway entry enzyme, E₁, may convert a commodity substrate to a product that is a selective substrate for one or a relatively small number of enzymes.

Competing enzymes utilize a substrate or product of the pathway of interest, which may include any one of P_(F), S₁, S₂, S₃, S₄, etc., and may be referred to as competing enzymes (E_(C)).

A specific non-limiting example of a pathway is the pathway for the synthesis of shikimic acid. For specific examples of this pathway, see FIG. 1 and FIG. 2. In this pathway, for example, a reaction between the cellular commodity compounds phosphoenolpyruvate (S_(1A)) and erythrose-4-phosphate (S_(1B3)) is catalyzed by the isoenzymes of DAHP synthase, aroFGH (E₁) to form 3-deoxy-D-arabinoseheptulose-7-phosphate (DAHP). DAHP (S₂) is transformed to 3-dehydroquinate (3-DHQ) by the second enzyme in the pathway, DHQ synthase, aroB (E₂). 3-DHQ (S₃) is dehydrated to 3-dehydroshikimate by the third enzyme in the pathway, 3-DHQ dehydratase, aroD (E₃). 3-dehydroshikimate (S₄) is reduced to shikimic acid (P_(F)) by the fourth enzyme in the pathway, shikimate dehydrogenase, aroE (E₄), using NADPH as a cofactor. The enzymes of the pathway are known in the art and have been characterized in a number of organisms, including, for example, E. coli, in which the enzymes are encoded by the genetic loci as follows: DAHP synthase (aroG, aroF); DHQ synthase (aroB); 3-DHQ dehydratase (aroD); shikimate dehydrogenase (aroE).

In the SA pathway, the overexpression of aroE, aroF, and tktA has been shown to increase the production of SA either directly with aroE and aroF, or indirectly with tktA, which increases the supply of the substrate molecule erythrose-4-phosphate. By protecting the mRNAs of enzymes that are rate-limiting, the pathway flux is increased dramatically.

There are also competing enzymes relevant to this pathway, which utilize the desired final product (P_(F)) as a substrate, which enzymes are shikimate kinase (aroK) and shikimate kinase II (aroL). The presence of active competing enzymes may be undesirable, as they reduce the amount of desired product from the reaction mixture.

Interferase.

As used herein, the term interferase refers to a microbial enzyme that is a site specific endoribonuclease (for a review, see Yamaguchi and Inouye (2009) Prog Mol Biol Trans) Sci. 85:467-500). Such enzymes may be one of an antitoxin-toxin pair.

In a preferred embodiment the interferase is a sequence specific endoribonuclease. Many such sequence specific endoribonuclease interferase enzymes are known to one of skill in the art, including, without limitation, the E. coli enzyme MazF and homologs thereof, which have been shown to be a sequence-specific (ACA) endoribonuclease that cleaves cellular mRNAs and effectively blocks protein synthesis (Zhang et al. (2005) J Biol Chem. 280(5):3143-50). ChpBK is a sequence-specific endoribonuclease that cleaves mRNAs both in vivo and in vitro at the 5′- or 3′-side of the A residue in ACY sequences (Y is U, A, or G) (Zhang et al. (2005) J.B.C. 280:26080-26088). MqsR is a GCU-specific mRNA interferase in E. coli (Yamaguchi et al. (2009) J Biol Chem. 284(42):28746-53)

In other embodiments the interferase, or a second interferase used in combination with a sequence specific endoribonuclease, is a site specific endoribonuclease. YafO is a ribosome-dependent mRNA interferase inhibiting protein synthesis (Zhang et al. (2009) J Biol Chem. 284(38):25522-31. RelE is a site specific endoribonuclease that cleaves mRNA at the A site of a ribosome.

For the purposes of the invention it may be desirable to introduce an exogenous interferase coding sequence into a microbial cell, operably linked to a regulatable promoter, e.g. an inducible promoter, which may be referred to herein as an interferase expression construct. The interfase expression construct may be provided on an episomal vector, e.g. a plasmid, YAC, BAC, viral, etc. vector, as known in the art. Alternatively the interferase expression construct may be integrated into the chromosome of the microbial host cell.

Interferase Cleavage Site.

As described above, a sequence specific interferase enzyme cleaves mRNA in a sequence specific manner, e.g. at GCU, ACA, ACY, etc. The methods of the invention utilize, in part, a modification of targeted genetic sequences to make the sequence of interest interferase resistant. The sequence of interest is altered by one or both of deletion of a residue in the motif for interferase cleavage, or by substitution of a residue in the motif with a neutral residue. By “neutral substitution” it is intended that the nucleotide at a position within the motif is substituted with a nucleotide that maintains the coding sequence or regulatory function, but which alters the sequence such that the motif is no longer present. A transcribed, i.e. mRNA sequence that is devoid of an interferase motif sequence may be referred to as resistant to that interferase.

In some embodiments of the invention, a plurality of pathway enzyme coding sequences are genetically modified to be interferase resistant, usually at least two coding sequences, at least three coding sequences, or more. Such modified coding sequence may be referred to as interferase resistant pathway enzyme sequences. The protected enzymes are usually rate-limiting enzymes in the pathway.

The use of the interferase resistant sequences allows protection of certain mRNAs, usually mRNA encoding rate limiting enzymes, thus increasing flux through a pathway of interest. The mRNAs of the pathway enzymes to be protected do not necessarily have to lie in the shortest path from substrate to product, as long as their overexpression leads to an increase in the flux to product. When the interferase is expressed, the mRNAs that code for most proteins in the cell are cleaved and degraded, but the proteins themselves can stay active for days. The interferase-resistant mRNAs, on the other hand, will not be degraded and the proteins that are coded by them will be overproduced upon activation of the interferase. This, in turn, results in an increased flux through the pathway of interest.

Site Specific Protease.

As used herein, the term refers to a protease, generally an endoprotease, which cleaves selectively at a specific amino acid motif, usually a motif of at least 4 amino acid residues to reduce background protein cleavage, and may be a motif of at least 5 amino acids, at least 6 amino acids, or more. Such proteases are known to those of skill in the art, and include, without limitation, tobacco etch virus protease (ENLYFQ^(G)/_(S)); yellow fever virus protease (GARR^(G)/_(S)); thrombin (LVPRGS); Factor Xa (I^(E)/_(D)GR), etc.

For the purposes of the invention it may be desirable to introduce an exogenous protease coding sequence into a microbial cell, operably linked to a regulatable promoter, e.g. an inducible promoter, which may be referred to herein as an protease expression construct. The promoter may be induced by an agent that is the same or different as the inducing agent for the interferase expression construct, usually different. The protease expression construct may be provided on an episomal vector, e.g. a plasmid, YAC, BAC, viral, etc. vector, as known in the art. Alternatively the protease expression construct may be integrated into the chromosome of the microbial host cell.

In some embodiments, optionally in combination with modification and expression of interferase resistant pathway enzyme sequences, the genetic sequence encoding the protease is modified to be interferase resistant, i.e. lacking any sequence motifs recognized and cleaved by the interferase of interest.

Protease Cleavage Site.

As described above, a sequence specific protease cleaves proteins in a sequence specific manner, e.g. at ENLYFQ^(G)/_(S), GARR^(G)/_(S), LVPRGS, I^(E)/_(D)GR, etc. The methods of the invention utilize, in part, a modification of targeted genetic sequences to make a targeted protein labile to protease digestion. In addition, the protease may be selected such that the pathway enzymes of interest are not cleaved by the protease. Alternatively the pathway enzymes of interest are modified such that the proteins are free of protease cleavage motifs.

The protein targeted for protease cleavage is usually a competing enzyme in the pathway of interest, i.e. an enzyme that utilizes a substrate or product of the pathway and by doing so reduces flux through the pathway. The targeted protein is altered by amino acid substitution to generate a cleavage site, preferably by a neutral substitution with conservative amino acid changes. By “neutral substitution” it is intended that the amino acid at a position within the motif is substituted with an amino acid that maintains the enzyme activity. Such an amino acid sequence may be referred to as protease labile sequence. In general the site for cleavage is selected such that, following cleavage, the enzyme activity is destroyed. See Example 2 herein.

“Cell-free system,” as used herein, is an isolated cell-free system containing a cell expressly engineered to synthesize an enzyme or cascade of enzymes that, when acting in a given sequence (e.g., in an enzymatic pathway) and proportion over a determined substrate, results in the preferential generation of a compound of interest. A compound of interest is typically a chemical entity (e.g., a small molecule), which can be used as an active pharmaceutical ingredient (API), chemical precursor, or intermediate, etc.

“Substrate,” as used herein, is a compound or mixture of compounds capable of providing the required elements needed to synthesize a compound of interest.

“Adenosine triphosphate regeneration system” or “ATP regeneration system,” as used herein is a chemical or biochemical system that regenerates AMP and ADP into ATP. Examples of ATP regeneration systems include those involving glucose metabolism, glutamate metabolism, photosynthesis, etc.

“Reducing equivalent,” as used herein, is a chemical species which transfers the equivalent of one electron in a redox reaction. Examples of reducing equivalents are a lone electron (for example in reactions involving metal ions), a hydrogen atom (consisting of a proton and an electron), and a hydride ion (:H—) which carries two electrons (for example in reactions involving NAD). A “reducing equivalent acceptor” is a chemical species that accepts the equivalent of one electron in a redox reaction.

Metabolite.

A metabolite is any substance produced during metabolism. For the purposes of the present invention, a metabolite is often, although not always, the product of an enzyme in the pathway of interest.

Inducible Expression.

The methods of the invention make use of regulated expression of various coding sequences, including without limitation the sequences encoding interferase and protease enzymes. Expression may be regulated by various cues, for example induction by chemicals, change of growth phase, depletion of a nutrient, temperature shifts, light, etc. In some embodiments inducible promoters regulated by the presence of an inducing agent, e.g. a chemical such as lactose, arabinose, tetracycline, etc., as known in the art.

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the coding sequence of interest. Promoters are untranslated sequences located upstream (5′) to the start codon of a structural gene that control the transcription and translation of particular nucleic acid sequence to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. At this time a large number of promoters recognized by a variety of potential host cells are well known. While the native promoter may be used, for most purposes heterologous promoters are preferred, as they generally permit greater transcription and higher yields.

Promoters suitable for use with prokaryotic hosts include the 3-lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and numerous hybrid promoters such as the tac promoter. However, other known bacterial promoters are also suitable, e.g. the lacI promoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter, the lambda PL promoter, promoters from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), and the acid phosphatase promoter. Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to a sequence of interest using linkers or adaptors. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the coding sequence.

Promoters suitable for eukaryotic cells, e.g. yeast cells, are also known in the art. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CXCAAT region where X may be any nucleotide. At the 3′ end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3′ end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors. Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglyceratekinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Yeast enhancers also are advantageously used with yeast promoters.

Production Methods

High yield synthesis of a product of interest is accomplished by providing a pathway system comprising pathway enzymes, substrates, co-factors and such salts, buffers, etc. as are required for enzyme activity, in a metabolically frozen cell or lysate thereof. The flux through the pathway and production of the final product is enhanced by genetically engineering the cell to over-produce rate-limiting enzymes in the pathway, and optionally to degrade enzymes that compete for substrates and/or products of the pathway. The cell is metabolically frozen at a desired growth point by inducing expressing of interferase activity, where the pathway enzymes or a subset there, e.g. rate-limiting pathway enzymes, have been modified to be interferase resistant. Modified enzymes may be indirectly related to the pathway of immediate interest if they increase the flux through the pathway. This procedure can be used to overcome regulatory (allosteric, transcriptional, post-transcriptional, etc.) or kinetic limitations. The effect of competing enzymes may be addressed by inducing expression of a sequence specific protease that cleaves one or more of the competing enzymes.

Induction of interferase activity may comprise upregulating expression of the interferase or downregulating expression of the cognate antitoxin for the interferase. The antitoxin may be native or exogenous to the cell, e.g. by expressing a siRNA that targets the antitoxin RNA, by including a protease tag on the antitoxin and express the cognate protease, by a temperature shift that shuts off an antitoxin promoter etc. Upregulation of interferase activity may comprise increasing the copy number of toxin, which would cause its overexpression even if induced from a promoter that is weaker than the native one; introducing mRNA stabilization signals such that the levels of toxin increase with respect to native one when induced; expression of the interferase from an inducible non-native promoter; expression of a non-coding RNA that either stabilizes the toxin, or destabilizes/silences the antitoxin; and the like.

The interferase coding sequence can be under the control of synthetic or native promoters, e.g. to activate the state of interest in response to internal or external signals. Cells can be metabolically frozen in any physiological stage, e.g. after exposure to certain stresses or growth conditions, and in any genotypic background, e.g. endonuclease-resistant sequences of different enzymes can be introduced or chromosomal deletions added, in order to bring about diversion of substrates that are normally used for growth towards pathways or products of interest. Cells of different genetic backgrounds, e.g. previously altered or genetically engineered, or species, or that are prepared by different strategies can be mixed and simultaneously or sequentially used in a bioprocess. The biocatalyst can be free or immobilized, and can be reused or disposed at each stage of the process.

The methods of the invention provide for high yields of the desired product, which yield is greater than the yield that can be achieved with a native microbial host. Productivity (i.e. rate of production per unit of volume or biomass) may also be increased. In one embodiment of the invention, the yield of product is at least about five-fold the basal rate, at least about 10-fold the basal rate, at least about 25-fold the basal rate, or more.

In some embodiments, interferase activity is induced by adding extracellular-death factor (EDF) to the medium, either naturally- or synthetically-derived. Spent medium can also be used to activate the dormant state.

The enzymes being expressed in metabolically-frozen cells can be harmful by themselves or through the enzymatic activities they have, or can be active under unfavorable process conditions, e.g. heat-resistant enzymes, osmotolerant enzymes, engineered enzymes, etc. Gene products that are not needed in the frozen state or that compete with pathways of interest but are essential for growth and thus cannot be deleted completely can be eliminated by modifying the coding sequence to be protease labile.

Different inocula can be adapted to different conditions (e.g. two batches grown on two different carbon sources) or can have different genotypes (e.g. different sets of enzymes made interferase-resistant, different enzymes made protease-labile, etc.) and then mixed to carry out the fermentation (e.g. to get simultaneous consumption of a mix of carbon sources or sequential processing of a metabolite through a pathway divided in two separate batches of cells). Fermentation can also take place sequentially by allowing one set of reactions to proceed in one vessel and then passing the supernatant through a second vessel. The toxin, antitoxin, or enzymes of the pathway of interest (or their mRNAs) can be engineered to manipulate their activity (e.g. by changing the specificity of the endonuclease targets) or otherwise optimized using molecular biology, biochemistry, or similar techniques. The methods can be practiced with intact cells or cell lysates.

The reactions may utilize a large scale reactor, small scale, or may be multiplexed to perform a plurality of simultaneous syntheses. Continuous reactions will use a feed mechanism to introduce a flow of reagents, and may isolate the end-product as part of the process. Batch systems are also of interest, where additional reagents may be introduced to prolong the period of time for active synthesis. A reactor may be run in any mode such as batch, extended batch, semi-batch, semi-continuous, fed-batch and continuous, and which will be selected in accordance with the application purpose.

The reactions may be of any volume, either in a small scale, usually at least about 1 ml and not more than about 15 ml, or in a scaled up reaction, where the reaction volume is at least about 15 ml, usually at least about 50 ml, more usually at least about 100 ml, and may be 500 ml, 1000 ml, or greater up to many liters of volume. Reactions may be conducted at any scale.

Various nutrients, salts, and buffers may be included, where ionic and nutrient species are typically optimized with regard to product production. When changing the concentration of a particular component of the reaction medium, that of another component may be changed accordingly. Also, the concentration levels of components in the reactor may be varied over time. The adjuster of oxidation/reduction potential may be dithiothreitol, ascorbic acid, glutathione and/or their oxidized forms.

In a semi-continuous operation mode, the reactor may be operated in dialysis, diafiltration batch or fed-batch mode. A feed solution may be supplied to the reactor through the same membrane or a separate injection unit. Synthesized product is accumulated in the reactor, and then is isolated and purified according to the usual method for purification after completion of the system operation.

Where there is a flow of reagents, the direction of liquid flow can be perpendicular and/or tangential to a membrane. Tangential flow is effective for recycling ATP and for preventing membrane plugging and may be superimposed on perpendicular flow. Flow perpendicular to the membrane may be caused or effected by a positive pressure pump or a vacuum suction pump or by applying transmembrane pressure using other methods known in the art. The solution in contact with the outside surface of the membrane may be cyclically changed, and may be in a steady tangential flow with respect to the membrane. The reactor may be stirred internally or externally by proper agitation means.

The amount of product produced in a reaction can be measured in various fashions. One method relies on the availability of an assay which measures the activity of the particular product being produced.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of the invention or to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, and the like), but some experimental errors and deviations may be present. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of the invention or to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, and the like), but some experimental errors and deviations may be present. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1 Interferase Resistant Pathway Enzymes

The sequences encoding enzymes from shikimic acid pathway enzymes are genetically modified to be interferase resistant by deletion of the ACA motif sequence recognized by mazF interferase. The sequences of Escherichia coli feedback-resistant DAHP synthase (encoded by aroG-fbr or aroF-fbr) are obtained (see Ger et al. (1994) J. Biochem. 116, 986-990; Kikuchi et al. (1997) Appl. Environ. Microbiol. 63, 761-762; and Weaver et al. (1990) J. Bacteriol. 172, 6581-6584). The sequence of the wild-type aroF is accessed at Genbank, NP_417092.1. The sequence of wild-type aroG is accessed at Genbank, NP 415275.1. The E. coli chromosome sequence is available in Genbank at accession number 000096, and provides the sequence for transketolase (tktA), and shikimate dehydrogenase (aroE).

The sequence corresponding to the mRNA for each of tktA, aroE, and aroG-fbr or aroF-fbr are scanned for the presence of ACA sequences. Each such motif is scored as being present in a coding sequence, a non-coding sequence with regulatory function, and non-coding sequence without regulatory function. For non-coding sequences without regulatory function, one of the nucleotides in the ACA motif is substituted with a different nucleotide. For non-coding sequences with regulatory function, one of the nucleotides can be substituted such that the regulatory function is not altered.

For coding regions, codon degeneracy is used to make changes, such that the amino acid sequence is not changed. Because of degeneracy, this can always be done whenever an A or C falls in the third base of the codon. Since the only amino acids with no degeneracy are methionine and tryptophan, but they do not have A or C in the third base, this does not present a challenge. Whenever more than one choice for codon change is possible, e.g. for arginine, codon usage information of the organism of interest, E. coli in this case, is taken into account to decide what substitution to make. After the modified, ACA-less sequence is figured out on paper, the DNA corresponding to the desired sequence is synthesized de novo. Alternatively the modifications are done in a cloned version of the gene using standard site-directed mutagenesis protocols, such as those for sale by Stratagene, e.g. Quickchange kit.

The modified sequences are introduced into a host. For initial testing E. coli is used as a host, although synthesis of other compounds may utilize other hosts, including, e.g., lactic acid produced in Lactobacillus spp. or Saccharomyces cerevisiae. The interferase-resistant versions of the genes can be either expressed from their native promoter or, alternatively, from the same inducible promoter from which the interferase is expressed. Preferably the chromosomal copy of the sequence is modified, so that no plasmids or promoter changes are required, although for some purposes a plasmid is used for episomal expression. In that case, the modified pathway enzyme coding sequences can be carried in the same plasmid, in a synthetic operon, or from more than one compatible plasmid, from their native promoters. Mutiple compatible plasmids can be introduced into the host microorganism.

Example 2 Protease Labile Competing Enzymes

The sequence encoding the competing enzyme, shikimate kinase I (aroK, Genbank accession number YP_026215.2) is inactivated by the methods set forth in Datsenko and Wanner (2000) Proc Natl Acad Sci USA. 97(12):6640.

The sequence encoding the competing enzyme, shikimate kinase II (Genbank accession number NP_414922.1) is made susceptible to proteolysis by a site-specific protease, by introducing a protease cleavage site in the target protein through introduction of specific mutations, by recombination, or multiplex genome engineering. The site for protease inactivation is introduced at a suitable site in the enzyme. Alternatively, shikimate kinase II is inactivated by the methods set forth in Datsenko and Wanner (2000), and shikimate kinase I is made susceptible to proteolysis by a site-specific protease.

The sequence of the enzyme is analyzed to identify the amino acid sequence closest to the protease cleavage motif, so that the least number of modifications to the amino acid sequence is needed to get a protease cleavage site. The ideal site is on the surface of the protein, to minimize the effects on enzyme function. A site near the surface also ensures that it is accessible to the protease when folded. Alternatively, an enzyme from a different organism that carries out the same function and contains a protease cleavage site can be used instead of a native enzyme that is not protease labile.

A suitable site for cleavage (a slash indicates alternative amino acids) is the sequence ENLYFQ^(G)/_(S) (cleaved by the tobacco etch virus protease). An alternative site for cleavage is GARR^(G)/_(S) (cleaved by the yellow fever virus protease). An alternative site for cleavage is LVPRGS (cleaved by thrombin). An alternative site for cleavage is I^(E)/_(D)GR (cleaved by thrombin). The genetically modified sequence, which may be referred to the protease-labile form of the sequence, is expressed from a plasmid with concomitant inactivation of the chromosomal copy, or used to alter the chromosomal sequence of the host organism. In an alternative embodiment, aroL is inactivated and aroK is made susceptible to proteolysis.

Enzymatic assays are conducted, after a brief scanning of the sequences, to ensure that the chosen protease cleaves the target protein, but not any other protein needed for SA biosynthesis. To accomplish this, the cells modified with the protease-labile competing enzyme are further modified to express the cognate protease under the control of an inducible promoter. The cells are grown in medium containing glucose as a carbon source. The production of shikimic acid in the cells is measured using H-NMR or HPLC, as described by Knop et aL (2001) J. Am. Chem. Soc. 123:10173-10182 or van Hess et al. (1999) Talanta, 5 Jan. 1999, Pages 173-17], or any other analytical chemistry technique, such as GC-MS.

Alternatively individual enzymatic assays, e.g., in which the depletion of an enzymatic substrate or accumulation of its product is measured in a solution with the purified enzyme protein, is performed.

If any protein in the SA pathway is found to be susceptible to proteolysis, other than the competing enzyme(s), cleavage sites are removed by making conservative amino acid changes by site-directed mutagenesis. Alternatively, an enzyme from a different organism that carries out the same function, but does not contain a protease cleavage site, can be used instead of a native enzyme that is protease labile. Such analysis is repeated if needed until an appropriate protease is found.

Example 3 Interferase Resistant Protease

The genetic sequence encoding the selected site-specific protease is modified to remove ACA sequences by site-directed mutagenesis, as described above in Example 1. The modified sequence is placed under the control of an inducible promoter. In one embodiment the promoter is the Pbad promoter, which is inducible with the inducing agent arabinose. The araE gene, which is responsible for arabinose transport, is optionally placed under a constitutive promoter in the host strain.

Example 4 Inducible Interferase Expression

The genetic sequence encoding the cognate interferase gene for the interferase-resistant pathway enzymes and protease is placed under an inducible promoter. A suitable interferase is mazF. The accession number for E. coli mazF is NP_417262.1. The sequence is operably linked to a promoter that is induced with an agent other than the inducer in Example 3. In one embodiment the promoter is the Plac promoter, which is inducible with IPTG. If the promoter leaks, causing the interferase gene to be induced early, extra copies of the repressor, lacI, are introduced into the host cell.

Example 5 Host Cell Growth

Cells incorporating the genetic changes set forth in Examples 1-4 are grown to a desired cell density in liquid medium. At the desired density the inducing agent for the interferase gene is added to the culture at a concentration sufficient to induce expression of the interferase. This induces a state of dormancy with concomitant overproduction of the feedback-resistant DAHP synthase, the transketolase, and the shikimate dehydrogeanse described in Example 1, causing an increased flux through the SA pathway. In this stage, where the pathway enzymes are being overproduced (or later when competing enzymes are cleaved), additional substrates may be required to be added to the medium, including amino acids, vitamins, buffers, etc.

To inactivate competing enzymes, the inducing agent for expression of the protease described in Example 3 is added to the cell culture. Induction of protease expression results in increased proteolysis of the labile shikimate kinase.

Example 6 Host Cell Lysate

The host cells of Example 5 are washed, and resuspended in suitable medium. The cells are lysed by homogenization. Glucose as a substrate for SA production is added to the medium, and synthesis of SA is measured to determine the production of this metabolite in the supernatant, using the assay described in Example 2.

Example 7

For synthesis of SA in intact cells, the host cells of Example 5 are washed and resuspended in suitable medium in the presence of glucose as a substrate. 

1. A cell genetically manipulated to comprise: an interferase coding sequence, which interferase cleaves mRNA at a specific sequence motif; coding sequences for at least two enzymes involved in a pathway of interest, wherein the coding sequences are modified to be free of motifs cleaved by said interferase.
 2. The cell of claim 1, wherein said interferase coding sequence is operably linked to an inducible promoter.
 3. The cell of claim 1, further comprising a coding sequence encoding a cognate antitoxin for said interferase.
 4. The cell of claim 1, wherein said at least two enzymes are rate-limiting enzymes in said pathway of interest.
 5. The cell of claim 1 wherein at least one of said enzymes is directly involved in said pathway of interest.
 6. The cell of claim 1, wherein at least one of said enzymes is indirectly involved in said pathway of interest.
 7. The cell of claim 1, wherein all the enzymes directly involved in the pathway of interest are modified to be free of motifs cleaved by said interferase.
 8. The cell of claim 1, wherein said interferase coding sequence is present on either an episomal vector, or a chromosome.
 9. The cell of claim 1, wherein the coding sequences for at least two enzymes involved in a pathway of interest are operably linked to an inducible promoter.
 10. The cell of claim 1, wherein the interferase is selected from MazF, ChpBK and MqsR.
 11. The cell of claim 1, wherein said pathway of interest is synthesis of shikimate or derivatives thereof.
 12. A cell genetically manipulated to comprise: a protease coding sequence operably linked to an inducible promoter, which protease cleaves proteins at a specific sequence motif; a coding sequence for at least one competing enzyme involved in a pathway of interest, wherein the coding sequence is modified to encode a motif cleaved by said protease.
 13. The cell of claim 12, wherein said protease coding sequence is present on either an episomal vector, or a chromosome.
 14. The cell of claim 12, wherein said pathway of interest is synthesis of shikimate or derivatives thereof.
 15. The cell of claim 12, further comprising: an interferase coding sequence operably linked to a second inducible promoter, which interferase cleaves mRNA at a specific motif; coding sequences for at least two enzymes involved in a pathway of interest, wherein the coding sequences are modified to be free of motifs cleaved by said interferase, and wherein the protease coding sequence is modified to be free of motifs cleaved by said interferase.
 16. A method of producing a product of a pathway of interest, the method comprising: growing a cell according to claim 1 to a desired population density; inducing expression of one or both of said interferase and said protease; providing said population of cells with substrates required for producing said product, wherein said enzymes in said pathway of interest catalyze production of said product.
 17. The method of claim 16, wherein said microbial cell is intact.
 18. The method of claim 16, wherein said microbial cell is lysed.
 19. A system for producing a product of a pathway of interest, the system comprising: a cell according to claim 1; and such substrate, nutrients, cofactors, buffers, reducing agents, ATP generating systems as are required for production of said product.
 20. The system of claim 19, wherein the cell is lysed. 